Superoxide dismutase catalyzes the conversion of superoxide radicals (O2xe2x88x92) into molecular oxygen (O2) and hydrogen peroxide (H2O2). The conversion of superoxide radicals is generally beneficial to a cell, since such molecules can react with the cell""s genomic DNA to induce mutations.
Superoxide dismutases (SOD) have been classified based on the inorganic atoms they require for activity. Three SOD families have been identified: those requiring manganese (MnSOD), those requiring iron (FeSOD), and those requiring copper and zinc (Cu, ZnSOD).
MnSODs have been found in mitochondria and prokaryotes, whereas FeSODs have been found in prokaryotes, primitive eukaryotes, and some plants. Cu, ZnSODs were originally found in eukaryotes and later found in several bacterial.
Macrophages are an important arm of a vertebrate""s immune system. Such cells can kill pathogens such as bacteria by engulfing the pathogen and bombarding it with superoxide radicals. Therefore, a secreted Cu, ZnSOD may play a role in the survival of bacterial pathogens, especially those known to survive and grow in macrophages.
The invention is based on the discovery of a secreted Cu, ZnSOD in Mycobacterium tuberculosis. It has been found that antibodies which specifically bind this M. tuberculosis SOD are useful in detecting the presence of the bacterium. It has also been discovered that tuberculosis patients develop antibodies against the M. tuberculosis Cu,ZnSOD. Thus, a patient producing antibodies against M. tuberculosis Cu,ZnSOD is diagnostic for tuberculosis in that patient.
Accordingly, the invention features an antibody, such as a monoclonal antibody, which specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, which is the amino acid sequence of the M. tuberculosis Cu,ZnSOD. Specific binding of an antibody to the polypeptide means that it does not substantially bind to other components within a sample. A Cu,ZnSOD or copper/zinc superoxide dismutase is a polypeptide that facilitates conversion of superoxide radicals to molecular oxygen and hydrogen peroxide, and whose superoxide dismutase activity is dependent on the presence of copper and zinc atoms or ions.
The invention also includes a method of detecting M. tuberculosis infection in a mammal by (1) providing a polypeptide comprising the amino acid sequence of SEQ ID NO:2; (2) contacting the polypeptide with a biological sample (e.g., a human serum sample) collected from the mammal, the contacting performed under conditions sufficient to allow an antibody to bind to the polypeptide; and (3) determining the presence of antibody bound to the polypeptide, wherein the presence of the antibody indicates M. tuberculosis infection in the mammal. This method optionally includes the step of removing antibodies which do not bind to the polypeptide.
The invention also features a method of testing whether a compound inhibits superoxide dismutase activity of a polypeptide by (1) contacting a polypeptide with the compound, the polypeptide being a Cu,ZnSOD (also called a copper/zinc superoxide dismutase) and having an amino acid sequence which is at least 50% (e.g., at least 60, 70, 80, 90 or 100%) identical to SEQ ID NO:2; (2) measuring the level of superoxide dismutase activity; and (3) comparing the level of superoxide dismutase activity in the presence of the compound with the level of superoxide dismutase activity in the absence of the compound. The compound is said to inhibit the superoxide dismutase activity of the polypeptide when the level of superoxide dismutase activity in the presence of the compound is lower than the level of superoxide dismutase activity in the absence of the compound. The polypeptide can be within a cell such as a bacterium, e.g., in the periplasm of the bacterium.
To facilitate the detection or testing methods of the invention, the polypeptide can be bound to a solid support (e.g., a plastic support such as a microtiter plate). In addition, the polypeptide can be covalently bound to a solid support bead such as Sepharose. The covalent linkage between the polypeptide and a support can be achieved by methods well known in the art. For example, the polypeptide can be covalently linked to a support by reacting it with chemically activated forms of the support (e.g., CNBr-activated Sepharose 4B or EAH Sepharose 4B, available from Pharmacia). In addition, equal amounts of the polypeptide can be deposited in each well of a microtiter plate, thereby creating an array on which multiple compounds can be tested in parallel or multiple samples can be assayed for the presence of M. tuberculosis. 
The invention relates to an antibody useful for detecting M. tuberculosis in a sample, methods of detecting M. tuberculosis infection in a mammal, and methods of testing a compound for its ability to inhibit SOD activity. These aspects of the invention arise from the discovery of a novel Cu,ZnSOD produced by M. tuberculosis. 
I. Polypeptides
The Cu,ZnSOD polypeptides useful in the methods of the invention include the M. tuberculosis Cu,ZnSOD polypeptide described below. The Cu,ZnSOD useful in the methods of the invention are not limited to the naturally occurring sequence. Cu,ZnSOD containing substitutions, deletions, or additions can also be used, provided that those polypeptides retain at least one activity associated with the naturally occurring polypeptide and are at least 50% identical to the naturally occurring sequence.
To determine the percent identity of two polypeptide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positionsxc3x97100).
The determination of percent homology and identity between two sequences can be accomplished using a mathematical algorithm. An example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc Natl Acad Sci USA 87:2264-2268 (1990), modified as in Karlin et al., Proc Natl Acad Sci USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol Biol 215:403-410 (1990). BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules useful in the methods of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers et al., CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences is determined using any of the above-described techniques with allowances for gaps. In calculating percent identity, only exact matches are counted.
An example of a Cu,ZnSOD that is not naturally occurring, though useful in the methods of the invention, is a Cu,ZnSOD-glutathione-S-transferase fusion protein. Such a protein can be produced in large quantities in bacteria and easily isolated via glutathione affinity column. The fusion protein can then be used in an in vitro SOD assay in the presence or absence of a candidate inhibitor of SOD (i.e., a candidate M. tuberculosis antimicrobial agent).
II. Antibodies
The term xe2x80x9cantibodyxe2x80x9d as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as M. tuberculosis Cu,ZnSOD. A molecule which specifically binds to M. tuberculosis Cu,ZnSOD is a molecule which binds M. tuberculosis Cu,ZnSOD, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains M. tuberculosis Cu,ZnSOD. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(abxe2x80x2)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind M. tuberculosis Cu,ZnSOD. The term xe2x80x9cmonoclonal antibodyxe2x80x9d or xe2x80x9cmonoclonal antibody compositionxe2x80x9d, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of M. tuberculosis Cu,ZnSOD. A monoclonal antibody composition thus typically displays a single binding affinity for the M. tuberculosis Cu,ZnSOD protein with which it immunoreacts.
Polyclonal antibodies against M. tuberculosis Cu,ZnSOD can be prepared by immunizing a suitable subject with a M. tuberculosis Cu,ZnSOD immunogen. The anti-M. tuberculosis Cu,ZnSOD antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized M. tuberculosis Cu,ZnSOD. If desired, the antibody molecules directed against M. tuberculosis Cu,ZnSOD can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-M. tuberculosis Cu,ZnSOD antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as ones described in Kohler et al., Nature 256:495-497, 1975; Kozbor et al., Immunol Today 4:72, 1983; and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985. The technology for producing various monoclonal antibody hybridomas is well known (see, e.g., Coligan et al., eds., Current Protocols in Immunology, John Wiley and Sons, Inc., New York, N.Y., 1994). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a M. tuberculosis Cu,ZnSOD immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds M. tuberculosis Cu,ZnSOD.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an antibody against M. tuberculosis Cu,ZnSOD (see, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977; Kenneth, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner Yale J. Biol. Med., 54:387-402, 1981). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (xe2x80x9cHAT mediumxe2x80x9d). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (xe2x80x9cPEGxe2x80x9d). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind M. tuberculosis Cu,ZnSOD, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody against M. tuberculosis Cu,ZnSOD can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with M. tuberculosis Cu,ZnSOD to thereby isolate immunoglobulin library members that bind M. tuberculosis Cu,ZnSOD. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP(trademark) Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690, WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum Antibod Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; and Griffiths et al., EMBO J 12:725-734, 1993.
Additionally, recombinant antibodies against M. tuberculosis Cu,ZnSOD, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication Nos. WO 87/02671 and WO 86/01533; European Patent Application Nos. 184187, 171496, 173494, and 125023; U.S. Pat. Nos. 4,816,567 and 5,225,539; Better et al., Science 240:1041-1043, 1988; Liu et al., Proc Natl Acad Sci USA 84:3439-3443, 1987; Liu et al., J Immunol 139:3521-3526, 1987; Sun et al., Proc Natl Acad Sci USA 84:214-218, 1987; Nishimura et al., Canc Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; Shaw et al., J Natl Cancer Inst 80:1553-1559, 1988; Morrison, Science 229:1202-1207, 1985; Oi et al., Bio/Techniques 4:214, 1986; Jones et al., Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J Immunol 141:4053-4060, 1988.
An antibody against M. tuberculosis Cu,ZnSOD (e.g., monoclonal antibody) can be used to isolate M. tuberculosis Cu,ZnSOD by standard techniques, such as affinity chromatography or immunoprecipitation. An antibody against M. tuberculosis Cu,ZnSOD can facilitate the purification of natural M. tuberculosis Cu,ZnSOD from the bacteria and of recombinantly produced M. tuberculosis Cu,ZnSOD expressed in host cells. Moreover, an anti-M. tuberculosis Cu,ZnSOD antibody can be used to detect M. tuberculosis Cu,ZnSOD protein (e.g., in a cellular lysate or serum sample) in order to evaluate the abundance of the M. tuberculosis Cu,ZnSOD protein. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, xcex2-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive material include 125I, 131I, 35S or 3H.
III. Contacting a Compound with a Cu,ZnSOD
For in vitro assays, contacting the compound with the Cu,ZnSOD can occur by mixing the Cu,ZnSOD with the compound in a solution, suspension, or gel. This solution, suspension, or gel is then subjected to a SOD assay.
For cellular assays, any Cu,ZnSOD polypeptide can be expressed in a cell if the cell does not already express Cu,ZnSOD, or overexpressed in the cell if the cell already expresses Cu,ZnSOD. Methods of expressing proteins in a cell are well known in the art.
If the Cu,ZnSOD resides within a cell, the compound can be delivered into the cell by methods well known in the art. If the compound is a membrane-permeable molecule, then the compound can be directly mixed with the cell, allowing contact between the Cu,ZnSOD and the compound. If the compound is not membrane permeable, as is expected for many macromolecules, the compound can be delivered into the cell by electroporation, or if it is a polypeptide, a nucleic acid or viral vector.
In addition, the cell can be an animal cell in vivo. Delivery of a compound to the cell can be accomplished by any route known in the art, including intravenous injection. Alternatively, a polypeptide compound can be administered by a nucleic acid or viral vector if delivery into the cell is desired.
IV. Superoxide Dismutase Assays
Assays for superoxide dismutase activity can be determined by any standard technique know in the art. See, for example the assays described in Beauchamp et al., Anal Biochem 44:276-287, 1971; and references therein.
Many of these assays rely on photoreduction of nitro blue tetrazolium (NBT), a process mediated by the production of superoxide radicals. Superoxide dismutase activity is reflected in any inhibition of the reduction of NBT. Exposure to an appropriate light source will turn NBT into a blue dye readily quantifiable by its absorbance at 560 nm. In the presence of a superoxide dismutase, however, photoreduction of NBT to a blue dye will be decreased or eliminated. Specific procedures based on this general concept are well known in the art. See, for example, the procedure described below.